Quantitative ZAP-70 assay

ABSTRACT

An quantitative ZAP-70 assay is provided, with ZAP-70+ and ZAP-70− controls, normal human blood controls, an improved antibody with better signal to noise ratio, and using the median MEFL that is calibrated using a standard curve.

PRIOR RELATED APPLICATIONS

Not applicable.

FEDERALLY SPONSORED RESEARCH STATEMENT

Not applicable.

FIELD OF THE INVENTION

The invention relates to an improved ZAP-70 prognostic assay based onflow cytometry. The assay is improved by being made quantitative, ratherthan qualitative—that is rather than providing a simple yes/no answerfor the ZAP-70 expression test, a numerical result for the level ofZAP-70 expression is provided. Improvements include the use of astandard curve of fluorescence per unit antibody; the use of a firstcontrol containing both ZAP-70+ and ZAP70⁻ cells, and the use of normalblood samples as assay controls, and finally, two cutoff values areprovided, rather than single cutoff. The assay is thus made considerablymore reliable than the prior art assays, and can be effectively used inpredicting the prognosis of chronic lymphocytic leukemia.

BACKGROUND OF THE INVENTION

Chronic lymphocytic leukemia (CLL) is a malignancy of bone marrow cells(lymphocytes) that is found mostly in older people. It affects about8,200 Americans each year, representing about 22-30% of all leukemiacases according to the National Cancer Institute. Many patients with CLLlive for a long time without treatment and survive many years afterdiagnosis. Others, however, have disease that rapidly progresses andleads to death within a few years.

The clinical problem has been how to distinguish between these twogroups of patients in order to treat each group appropriately. Since thelate '90 s it has been known that the presence or absence of somaticmutations in the immunoglobulin heavy chain variable (IgVH) regions ofCLL cells provides a reliable distinguishing characteristic betweenthese two patient populations. Patients whose CLL cells expressun-mutated IgVH regions usually have early progression of their disease,whereas patients with mutated IgVH regions usually have laterprogression.

However, one major shortcoming of using IgVH mutations as a biomarkerfor CLL is that most clinical laboratories are not capable of detectingIgVH mutations. Even if readily available, the technology is expensiveto implement and time consuming, making it impractical for widespreadscreening of all CLL patients. Thus, scientists have been searching fora reliable surrogate marker for IgVH mutation.

Rosenwald found such a marker. In 2001 his group reported thatZeta-associated protein of 70 kDa (ZAP-70), a cytoplasmic tyrosinekinase essential for T-cell-receptor signal transduction, ispreferentially expressed in CLL B-cells whose immunoglobulin genes havenot undergone somatic hypermutation, while a second CLL subtype withmutated immunoglobulin genes most often lacks ZAP-70 expression.Rosenwald (2001); US2003203416.

Patients with less ZAP-70 in their B-cells were more likely to havemutations, and lived anywhere from 15 to 34 years after their diagnosis,with an average survival of 24.4 years. On the other hand, CLL patientswhose cells contained significant ZAP-70 were less likely to havemutations and lived from 7 to 11.5 years, with an average of 9.3 years.Recently, it was shown that ZAP-70 expression in T-cells correlates withB-CLL ZAP-70 and bears the same correlation with time to clinicalprogression of the disease (Herishanu 2005).

The use of ZAP-70 as a marker for IgVH mutation was initially verypromising, however, subsequent studies have cast doubt on its prognosticvalue. Researchers have criticized the current ZAP-70 assays, asfollows:

-   -   However, for both CD38 and ZAP-70, subsequent studies have        yielded controversial results with regard to their validity as a        surrogate marker for VH and prognostic indicator. The facts        that (1) divergent results have been obtained in different        laboratories    -   (CD38 and ZAP-70), (2) the expression level may change over time        (CD38), (3) a careful separation of T-cells is necessary        (ZAP-70), (4) different cut-off values to distinguish “positive”        from “negative” cases were defined (CD38 and ZAP-70), and (5)        approximately 10%-30% of cases show discordant status for CD38        or ZAP-70 as compared to VH in all series described, indicate        that these markers may not be as reliable as initially thought        for routine diagnostics. Byrd (2004) (cites omitted) (emphasis        added).

The National Cancer Institute has also noted that the test is notstandardized: “This test, which is commercially available, but has notundergone national standardization, has been proposed as a surrogate forthe mutational status.” Our own lab has noted that ZAP-70 is presentonly in low copy number against a large background of noise, and thesefacts have hindered the realization of the true prognostic value of theZAP-70 assay. Thus, it is apparent that the use of the ZAP-70 assay as aprognostic tool needs considerable improvement. What is needed in theart is a standardized assay with increased reliability, sensitivity andgreater quantitative precision.

SUMMARY OF THE INVENTION

The following abbreviations are used in herein: TABLE 1 AbbreviationsAbbreviation Expansion ABC Antibody Binding Capacity - The ABC is thenumber of monoclonal antibodies a sample will bind, and correlates tothe number of antigens expressed on the cell surface. Eff. F/P Theeffective number of fluorochrome molecules conjugated per each antibodymolecule determined by measured fluorescence intensity of antibodycapture microspheres measured on a flow cytometer calibrated in units offluorochrome specific MESF F/P Number of fluorochrome moleculesconjugated per each antibody molecule determined by absorbance on aspectrophotometer. FCS Fetal Calf Serum FSC Forward angle light scattermAb Monoclonal antibody MESF Molecules of Equivalent SolubleFluorochrome. Corrects for changes in extinction coefficient, quenching,and small spectra shifts. Using the appropriate calibration controls,MESF and ABC can be calculated directly by the software that controlsthe cytometer. See also Molecules of Equivalent Soluble Fluorochrome orMEFL, which is used interchangeably herein. MFI Mean FluorescenceIntensity pAb polyclonal antibody PB Peripheral blood PBS phosphatebuffered saline (200 mg/l KCl; 200 mg/l KH2PO4; 8 g/l NaCl; 2.16 g/lNa2HPO4•7H2O, pH 7.4) PermiFlow Working Solution A 1:1 mixture ofPermiFlow and Sigma water RNAse DNAse free

“ZAP-70⁺” or “ZAP-70 positive” cells are defined as cells having asignificant and known level of ZAP-70 expression. “ZAP-70⁻” or “ZAP-70negative” cells have a much lower level of ZAP-70, but need not becompletely devoid of ZAP-70 expression.

The routine, clinical determination of ZAP-70 expression in CLL hasproved problematic in many laboratories. Poor precision, caused by thelow copy number and a high level of non-specific binding, is asignificant issue in connection with the assay. Accordingly, we havedeveloped a quantitative assay that is standardized and provides betterresults.

Prominent in the literature (Crespo, 2003, Rassenti 2004) is the use ofthe tail of the distribution of ZAP-70 in normal or patient T-cells todefine positivity. Instead, the invention uses a bead standard curve toconvert MFI to MEFL using the medians of the distributions. The medianis not dependent on the shape of the distribution and so has betterprecision. In this way we have shown that the prior art approachescontribute significantly to poor precision. We have also developed aWinlist protocol that helps to standardize the data analysis.

Extensive quality control measures are also necessary to assayprecision. In addition to a daily normal peripheral blood control, theinvention uses a cultured cell line system containing cells with knownZAP-70 levels. An on-going review of patient data for the relativenumber with a negative result is also useful. It is emphasized thatmeticulous laboratory technique, including the strict standardization ofpipetting, incubation, and washing are of paramount importance toobtaining a reproducible assay.

In a preferred embodiment, the invention employs an Alexa fluor® 647conjugated anti-ZAP-70 antibody, rather than the more commonly used PEreagent, because of the better signal-to-noise ratio. In a furtherpreferred embodiment, the patients samples are fresh blood, becauseblood storage decreases apparent ZAP-70 levels (Letestu 2005). Agedsamples should be corrected for age-dependant reduction in ZAP-70levels.

We analyzed 375 patients using the NCCLS (CLSI) rank-order,non-parametric approach to reference ranges to assign a cutoff for“negative.” This cutoff was constructed at the 55th percentile of thepatients studied. The next 15% of the rank-order were designated as“intermediate.” The 35% with the highest values are, then, considered tobe positive. The use of the two-cutoff rather than a single cutofffurther improves the reliability of the assay.

Generally speaking the invention is a quantitative ZAP-70 assaydescribed as follows: stain a first control cell population with ananti-ZAP-70 antibody, an anti-T-cell antibody and an anti-B-cellantibody, wherein said first control cell population comprises mixtureof ZAP-70⁺ T-cells and ZAP-70⁻ B-cells. In preferred embodiments, thefirst control cell population can be a mixture of Jurkat (a T-cell line)and Ramos cells (a B-cell line). In another preferred embodiment, asecond control cell population comprising normal human blood can also beused. Measure anti-ZAP-70 antibody in the B-cells and T-cells byconverting mean fluorescent intensity (MFI) of anti-ZAP-70 antibody tomolecules of equivalent fluorochrome (MEFL) using a standard curve, andusing the median of the population as the ZAP-70 value. Confirm that theamount of ZAP-70 in the first control cell population is withinpre-established normal parameters, and if so, repeat on a patientsample, wherein if the level of ZAP-70 in the patient B-cells is in thetop 30% indicates that the patient has progressive chronic lymphocyticleukemia (CLL), and if the level of ZAP-70 in the patient B-cells is inthe lower 55% indicates that the patent has indolent CLL. In anotherembodiment, the level of ZAP-70 in T-cells can also be used, sinceT-cell ZAP-70 also correlates with time to progression of disease. AnAlexa fluor® 647 stain is particularly preferred. The T-cell antibodycan be anti-CD5 antibody, and the anti-B-cell antibody can be anti-CD19antibody, but many of the T- and B-cell antibodies are known andavailable.

Several anti-ZAP-70 antibodies are available, some of which are alreadyconjugated to fluorescent probes. Those antibodies that are notconjugated can easily be conjugated with an appropriate dye using one ofthe many available conjugation kits. Commercial suppliers will alsoprovide custom antibodies on demand. Exemplary antibodies include thefollowing: TABLE 2 Anti-ZAP-70 antibodies BD Biosciences #612588 MouseAnti-ZAP-70 Kinase mAb, FITC Conjugated, Clone 29 Pharmingen ™ MouseAnti-ZAP-70 Kinase mAb, Unconjugated, Clone 24a #612719 MouseAnti-ZAP-70 Kinase Monoclonal Antibody, Unconjugated, Clone 24a #612589Mouse Anti-ZAP-70 Kinase Monoclonal Antibody, FITC Conjugated, Clone 29#612718 Mouse Anti-ZAP-70 mAb, Unconjugated, Clone 29 #557818 MouseAnti-Zap70 (Y319)/Syk (Y352) mAb, Alexa Fluor ® 488 Conjugated #557817Mouse Anti-Zap70 (Y319)/Syk (Y352) Mab, Alexa Fluor ® 647 Conjugated#557881 Mouse Anti-Zap70 (Y319)/Syk (Y352) mAb, Phycoerythrin ConjugatedBiocare #CM259A Mouse Anti-ZAP-70 Research Use Only mAb, UnconjugatedMedical ™ BioLegend ™ #313403 Mouse Anti-ZAP-70 mAb, R-PhycoerythrinConjugated, Clone 1E7.2 # 313401 Mouse Anti-ZAP-70 mAb, Unconjugated,Clone 1E7.2 CalTag Lab. ™ #MHZAP7000 Purified anti-human mouse ZAP-70#MHZAP7020 Alexa Fluor 488 anti-human mouse ZAP-70 #MHZAP7004 R-PEanti-human mouse ZAP-70 #MHZAP7018 Pe-Cy5.5 anti-human mouse ZAP-70 CellSignaling #2701S Anti-Zap-70, phospho (Tyr319)/Syk, phospho (Tyr352)Antibody, Technology ™ Unconjugated #2704S Anti-Zap-70, phospho (Tyr493)Antibody, Unconjugated #2704L Anti-Zap-70, phospho (Tyr493) Antibody,Unconjugated #2707 Rabbit Anti-Zap-70 mAb, Alexa Fluor ™ 647 Conjugated,Clone 136F12 #2705 Rabbit Anti-Zap-70 mAb, Unconjugated, Clone 99F2Chemicon ™ #AB1377 Anti-ZAP-70 (70 kDa zeta-associated protein) pAb,Unconjugated Diagnostic #Mob 431 Anti-ZAP-70 mAb, Unconjugated, Clone2F3.2 BioSystems ™ eBioscience ™ #14-6695 Affinity Purifiedanti-mouse/human ZAP-70 (ZAP70) #13-6695 Biotin anti-mouse/human ZAP-70(ZAP70) #11-6695 FITC anti-mouse/human ZAP-70 (ZAP70) #12-6695 PEanti-mouse/human ZAP-70 (ZAP70) #35-6695 Phycoerythrin-Cy5.5 (PE-Cy5.5)anti-mouse/human ZAP-70 (ZAP70) GeneTex ™ #GTX73804 ZAP-70 Mouse mAb#GTX74255 ZAP-70 Rabbit pAb #GTX12868 ZAP70 (phospho Y292) Rabbit pAb#GTX12869 ZAP70 (phospho Y315 + Y319) Rabbit pAb #GTX13957 ZAP70 RabbitpAb #GTX18384 ZAP70 Mouse mAb #GTX18371 ZAP70 Mouse mAb #GTX14827 ZAP70Mouse mAb Novus #ab14827 Mouse Anti-ZAP70 mAb, Unconjugated, Clone 2F3.2Biologicals ™ #ab13957 Rabbit Anti-ZAP70 Pab, Unconjugated #ab12868Rabbit Anti-ZAP70, phospho (Tyr292) Pab, Unconjugated #ab12869 RabbitAnti-ZAP70, phospho (Tyr315/Tyr319) Pab, Unconjugated OriGene ™#TA201071 Zap-70 (99F2) Rabbit mAb Serotec Inc #AHP470 Rabbit Anti-HumanZAP-70 Pab, Unconjugated Spring #E4634 Rabbit Anti-ZAP-70 Pab,Unconjugated Bioscience ™ United States #Z0300 Anti-Human ZAP 70 mAb,Unconjugated, Clone 0.T.155 Biological ™ Upstate ™ #06-271 Anti-ZAP-70#05-776 Anti-ZAP-70, clone 1E7.2 #16-211 Anti-ZAP-70, clone 2F3.2,biotin conjugate, human #16-210 Anti-ZAP-70, clone 2F3.2, FITCconjugate, human #05-253 Anti-ZAP-70, clone 2F3.2, human #05-253MGAnti-ZAP-70, clone 2F3.2, human #16-214 Anti-ZAP-70, clone 2F3.2,(TRITC) Rhodamine conjugate, human Zymed Anti-ZAP-70 mAb, Unconjugated,Clone ZAP-70-6F7 Tebu-Bio ™ #021MHZAP7004 Anti ZAP-70 Human PE#021MHZAP7020 Anti ZAP-70 Human Alexa 488

The fluorescent labels should be chosen with the operatingcharacteristics of the cytometer in mind such that there is sufficientseparation of signal so as to allow the cytometer to distinguish betweenthe two or more signals. Many such labels are known in the art,including fluorescent isothiocyanate (FITC, aka fluorescein),Phycoerythrin (PE); Cy5PE; Cy7PE; Texas Red (TR); Allophycocyanin (APC);Cy5; Cy7APC; Cascade Blue; and the like. However, preferred labels arethe Alexa Fluor® labels available from Invitrogen™, CalTag™, BDBiosciences™, etc., including Alexa Fluor® 350, 405, 430, 488, 500, 514,532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700 and 750 dyes.Particularly preferred is Alexa Fluor® 647.

In order to detect an intracellular target such as ZAP-70, cells must befixed and permeabilized to allow the antibody to penetrate the cell andbind the antigen, while retaining sufficient cell morphology to allowcell separation. Thus, cells are permeabilized with agents such as 0.05%Triton X-100 in PBS, stained with antibody and then fixed. For example,PERMEAFIX™ or PERMIFLOW™ at mild denaturation or room temperaturespreserves cell morphology and thus allows subsequent analysis by flowcytometry. Such methods are described in more detail in co-pendingapplication 60/512,834 filed Oct. 20, 2003.

“Fixatives” are defined herein as fixatives that allow both cellfixation and permeation, while retaining cell surface morphology and DNAand RNA content, sufficient to allow separation of cells based on lightscatter, surface epitopes and/or nucleic acid content. An acceptablefixative contains 0.75-0.85% formaldehyde, 25-30 mM DNBS, 6.8-7% DMSOand 0.08-0.1% Tween 20 detergent. A preferred fixative is PERMIFLOW.™

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Standard Curve—Conversion of Mean Channels to MEFL. Using thesame standardized cytometry set-up in which patients are analyzed, aeight peak fluorescent standard is used to convert known equivalentfluorescent levels to linear mean channels (only six points shown in thegraph). The derived slope and intercept are used to convert the MFI toMEFL.

FIG. 2. Comparison of Fluorochrome Performance. Signal to noise ratio isplotted against antibody concentration for both Alexa fluor® 647 and PElabeled antibodies. Alexa fluor® 647 provides significantly improvedsignal to noise ratio, especially at lower concentrations. Cells werestained with the same primary antibody to SMAD-2 developed withsecondary antibody conjugated to either Alexa 647 or PE and at thevarious concentrations.

FIG. 3. Anti-ZAP-70 PE vs. Anti-ZAP-70 Alexa fluor® 647. Separation ofcontrol cell populations—the ZAP-70 negative Ramos cell line and theZAP-70 positive Jurkat cell line—is greatly improved with the Alexafluor® 647 label. This figure also illustrates the usefulness of thesecell lines as positive and negative controls. This figure is predictedfrom the data presented in FIG. 2 and holds true in the detection ofZAP-70.

FIG. 4. Normal Peripheral Blood. The cells in blue represent T cells,the cells in red represent B cells and the cells labeled green are NKcells. The data has been converted to MEFL as evidenced by the median Xvalues in the box. The single parameter histogram in the lower leftdemonstrates the ability to independently measure ZAP 70 levels in thevarious subsets.

FIG. 5. MEFL Levels of T-cells in Normal Subjects vs. CLL Patients.FIGS. 4 and 5 are the same data represented in different ways; FIG. 4 isa FACS scatter plot, and FIG. 5 is the calculated MEFL for the variouscell populations( 19th Annual CCS meeting,Abs#12.p.16,2004). TheRassenti (2004) and Crespo (2003) papers use T-cell levels for ZAP-70 toset the threshold level for positivity in the CLL cells. As this datashows, the prior art assays lack precision because their one-point“standard” is a moving target.

FIG. 6. Cutoffs—One Cutoff vs. Two. The prior used a single cutoff valueto assign patients as ZAP-70 positive or negative (FIG. 6 a). However,subsequent research has shown that this is too simplistic and a twocutoff assay is proposed in FIG. 6 b. Here, patients with high ZAP-70levels (greater than 2,100 MEFL) are positive, low ZAP-70 (less than1350 MEFL) are negative, but those between 1,350 and 2,100 MEFL ZAP-70are indeterminate.

FIG. 7. Multiple Site data. Showing that the assay is reproducibleacross different laboratories.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention is exemplified with respect to CLL. However, itcan be used for any disease for which ZAP-70 is a marker, including forexample, severe combined immuno-deficiency (SCID) (Otsu 2002), arthritis(Sakaguchi 2003), or other types of lymphoma (Admirand 2004).

The following examples describe the invention as practiced in additionaldetail, but should not be construed as limiting.

EXAMPLE 1 Materials

The sample can be any bone marrow or peripheral blood sample that isreceived in ACD, Sodium Heparin, or EDTA. In a preferred embodiment thesample is a fresh heparin sample. In addition to standard laboratoryequipment, the following was used herein: TABLE 3 Equipment and reagentsCytometer with filters for FITC, PE, Alexa fluor ® 647, and Alexafluor ® 700. anti-ZAP-70 Alexa fluor ® 647 (Cell Signaling Technology#2705) Buffer QC (1.0 M NaCl, 50 mM MOPS pH 7.0, 15% isopropanol (v/v))Microsoft ™ Excel 2000 ™ Newborn Calf Serum PBS + 2% NCS PermiFlow(Invirion ™ Corp. # 55001, Reagent #1) PermiFlow Working Solution (1:1mixture of PermiFlow ™ and Sigma ™ water RNAse/DNAse free) Rainbowcalibration particles (Spherotech, ™ Inc., Cat. #RCP-30-5A) RPMI(Invitrogen ™ #11875119) Surface Antibody Cocktail containinganti-CD5-FITC and anti-CD19-PE Water (Sigma ™ #W4502) WinList ™ Version5.0 (Verity Software House ™)

TABLE 4 Antibodies Cell Type Antibody Source T-cells anti-CD5-FITCImmunotech IM#0468 B-cells anti-CD19-PE Immunotech IM#1285 T-, B-, andNK-cells anti-ZAP-70-Alexa Cell Sig. Tech. Cat # 2707 fluor ® 647 T-,B-, and NK-cells anti-ZAP-70-PE Caltag ™ Catalog #MHZAP7004

EXAMPLE 2 Sample Preparation

About one million WBC cells/ml are needed for each patient sample. Theantibodies employed are shown in Table 4, above.

Deliver 100 μl of adjusted blood sample to a 12×75 mm tube. Add titeredamounts of Surface Antibody Cocktail or if cocktail is unavailable, usetitered amounts of anti-CD5 FITC and anti-CD19 PE antibodies to preparea “hand-made” cocktail. Vortex to mix. Allow to incubate in the dark for20 minutes.

Wash tubes in 4 ml of PBS+2% NCS. Spin at 1300 rpm for 5 minutes,decant, and blot. Add 4 ml of prepared PermiFlow Working Solution toeach tube and pipet up and down until there is no RBC pellet adhered tobottom of tube. Incubate at RT for 1 hour or at 4° C. overnight if testis to be completed the next working day.

Spin tubes to a pellet, decant, and add 4 ml of PBS+2% NCS, mix wellwith pipet. Spin to a pellet and repeat. If there is still a largeamount of residual lysed red cell debris in the bottom of tube asindicated by a solid red pellet, repeat wash until residual lysed redcell debris is removed.

Add saturating, titered amounts of the anti-ZAP-70 Alexa 647 to eachtube. Vortex to mix and incubate at RT for 1 hour. Wash twice in PBS+2%NCS. Re-suspend cells in 750 ul PBS+2% NCS.

Prepare Rainbow™ beads by adding 1 drop of vortexed Rainbow Beadsuspension to 1 ml of Buffer QC. Collect Rainbow Bead data.

EXAMPLE 3 Flow Cytometry

We used the BECKMAN COULTER™ FC500 or BECTON DICKINSON™ FACS CALIBURFLOW cytometer herein, but any properly quality controlled flowcytometer that satisfies established windows of analysis can be used,provided the laser excitation lines and filter configurations arecorrect for the excitation and detection of all fluorescence labels usedin the assay. In general, we collect 50,000 ungated events. Data wasanalyzed in WinList (VERITY SOFTWARE™) and Excel (MICROSOFT™) orequivalent. Exemplary results are shown in FIG. 1 through 6.

EXAMPLE 4 Antibody Optimization

One of the difficulties with the current assay is the ZAP-70 isexpressed at only low levels and the signal to noise ratio with existingantibodies is poor. Thus, to fully optimize the assay, we also studiedthe fluorescent behavior of multiple fluorochromes. We assessed theperformance of different fluorochromes at identical antibodyconcentrations. The graph in FIG. 2 depicts a typical finding comparingthe SMAD-2 primary antibody developed with secondary antibodiesconjugated to either PE or Alexa fluor® 647 using the same flowcytometer. The graph teaches us three things: 1) In ranges of 0.5-2 μg,Alexa fluor® 647 exhibits better signal to noise performance, and 2)there is a point where more antibody is not better, and 3) excess Alexafluors are more easily removed from the cell in comparison to PE.

We then tested these labels in a real cell separation experiment usingour Ramos/Jurkat controls (described in more detail below). We comparedthe anti-ZAP-70 rabbit monoclonal clone 136F12 conjugated with Alexafluor® 647 to a commercially available anti-ZAP-70 conjugated with PE.FIG. 3 shows greatly improved signal separation of the Alexa fluor®based label, and it was chosen for all future experiments.

We also optimized the level of each antibody. Antibody titration datawas plotted to show 1) staining intensity versus antibody stainingconcentration, 2) percent positive staining versus concentration and 3)the signal to noise ratio versus concentration (data not shown). Theseplots aided in the identification of the optimal staining concentrationof each antibody, indicated whether saturation staining was possible foreach antibody. The minimal antibody that provided saturation stainingwas chosen for future experiments.

EXAMPLE 5 Assay Design

An improved ZAP-70 assay should meet one of more of these basiccriteria: 1) the assay should be adaptable to peripheral blood, bonemarrow, and cell line systems; 2) the assay should be multiparametricwith a preferred minimum of 6 parameters(FSC,SSC,FITC/PE/Alexa647/Alexa700) combining surface/cytoplasmicstaining; 3) The assay should collect data uncompensated to removehardware bias (technologists who enter hardware values incorrectly canbias quantitative values versus off-line software compensation); 4) Theassay should convert fluorescence parameters to MEFL and quantitate allfluorescence parameters; and 5) the assay should measure each cellpopulation independently. Therefore, we designed a ZAP-70 assay to meetthese needs.

Our ZAP-70 assay requires the mean fluorescence intensity (MFI) to beconverted to molecules of equivalent fluorochrome (MEFL). MEFL providesa more precise indicator of ZAP-70 expression because it equates themedian intensity of the populations relative to a known, stablestandard. This is done using an 8 peak fluorescent standard to correlateknown equivalent fluorescent levels to linear mean channels. The slopeand intercept are derived by linear regression and used to convert themedian intensity to MEFL.

To further improve our assay we have added two types of quantitativecontrols. One of our controls is a mixture of equal numbers of Jurkatand Ramos cells. Jurkat cells express significant ZAP-70, while Ramoscells express much less ZAP-70. Thus, this mixture of cells providesuseful positive and negative controls for the assay (see e.g., FIG. 3).This material controls for cell fixation, permeation, antibody stainingand ZAP-70 expression levels across different laboratories andequipment. It also accounts for proper removal of excess antibodybecause elevated levels in the Ramos cells indicate poor lab technique.

We also employ a second kind of semi-quantitative control, using bloodfrom normal subjects. About 100 μl of blood is stained from a normalsubject, and this blood provides ZAP-70 positive NK and T-cells, as wellas ZAP-70 negative normal B-cells. The normal blood thus controls forcell separation, as well as fixation, permeation and staining, whileproviding three levels of ZAP 70 expression.

EXAMPLE 6 Evaluation

Finally, based on the available data, it is apparent that a simplesingle cutoff system for presence or absence of ZAP-70 is too simplisticand does not accurately reflect the biology of CLL. Instead we propose atwo cutoff system, providing three ranges ZAP-70 expression—positive,negative, and an in-between, indeterminate region where ZAP-70expression is inconclusive. Under the conditions described herein, wehave set the cutoff values at 2,100 MESF (30% positive), and 1,350 MESF(55% negative) with the intervening region being 15% indeterminate.Studies of patient samples show this grouping indicates our quantitativeassay matches the published distribution of patients with known IgVHstatus. TABLE 5 Data Normal Ranges: B-cells: 300-900 median MESFT-cells: 4300-9000 median MESF Patient: Negative ≦1350 Intermediate =1,350-2,100 Positive ≧2100*These ranges were determined in-house and may be subject to change dueto ongoing evaluation.

Further the assay was repeated at two locations to determine whether iswas reproducible across sites. The data in FIG. 7 shows that the assayis very reproducible.

The following articles are incorporated by reference in their entirety:

Admirand J. H. et al., Immunohistochemical detection of ZAP-70 in 341cases of non-Hodgkin and Hodgkin lymphoma, Mod Pathol. 17(8):954-61(2004)

Byrd J C, et al., Chronic lymphocytic leukemia, Hematology (Am SocHematol Educ Program) 1:163-83 (2004).

Crespo M., et al., ZAP-70 expression as a surrogate forimmunoglobulin-variable-region mutations in chronic lymphocyticleukemia. N Engl J Med 348 (18): 1764-75 (2003).

Herishanu Y., et al., Quantitative flow cytometry of ZAP-70 levels inCLL using molecules of equivalent soluble fluorochrome: B-CLL cell andT-cell ZAP-70 levels correlate with disease progression, Leukemia &Lymphoma 46(S1): S50 (2005).

Letestu R., et al., Evaluation of ZAP-70 expression by flow cytometry inchronic lymphocytic leukemia: a multicentric international harmonizationprocess, Leukemia & Lymphoma 46(S1): S47 (2005).

Orchard J A, et al., ZAP-70 expression and prognosis in chroniclymphocytic leukaemia. Lancet 363 (9403): 105-11 (2004).

Makoto Otsu, et al., Reconstitution of lymphoid development and functionin ZAP-70-deficient mice following gene transfer into bone marrow cells,Blood, 100(4): 1248-1256 (2002).

Rassenti L Z, et al., ZAP-70 compared with immunoglobulin heavy-chaingene mutation status as a predictor of disease progression in chroniclymphocytic leukemia. N Engl J Med 351 (9): 893-901 (2004).

Rosenwald et al., Relation of Gene Expression Phenotype toImmunoglobulin Mutation Genotype in B-cell Chronic Lymphocytic Leukemia,The Journal of Experimental Medicine, 149:1639-1647 (2001).

Sakaguchi N, et al., Altered thymic T-cell selection due to a mutationof the ZAP-70 gene causes autoimmune arthritis in mice, Nature.426(6965):454-60 (2003).

1) A quantitative ZAP-70 assay, comprising the steps of: a) staining afirst control cell population with an anti-ZAP-70 antibody, ananti-T-cell antibody and an anti-B-cell antibody, wherein said firstcontrol cell population comprises mixture of ZAP-70⁺ T-cells and ZAP-70⁻B-cells; b) measuring anti-ZAP-70 antibody in the B-cells and T-cells byconverting mean fluorescent intensity (MFI) of anti-ZAP-70 antibody tomolecules of equivalent fluorochrome (MEFL) using a standard curve, andusing the median of the population as the ZAP-70 value; c) repeatingsteps i)-iv) on a patient blood sample if the amount of ZAP-70 in thefirst control cell population is within pre-established normalparameters, wherein a level of ZAP-70 in the patient B-cells in the top30% indicates that the patient has progressive chronic lymphocyticleukemia (CLL), and a level of ZAP-70 in the patient B-cells in thelower 55% indicates that the patient has indolent CLL. 2) The assay ofclaim 1, wherein the first control cell population is a mixture ofJurkat and Ramos cells. 3) The assay of claim 2, further comprisingrepeating steps i)-iv) using a second control cell population comprisingnormal human blood. 4) The assay of claim 3, wherein anti-T-cellantibody is anti-CD5 antibody, and the anti-B-cell antibody is anti-CD19antibody. 5) The assay of claim 4, where the anti-ZAP-70 antibodycomprises an Alexa fluor® 647 stain. 6) The assay of claim 5, whereinthe B-cells and T-cells are electronically separated by flow cytometry.7) The assay of claim 6, wherein ≧2100 MEFL in the patient B-cellsindicates said patient has progressive CLL, and ≦1350 MEFL in thepatient B-cells indicates said patient has indolent CLL. 8) Aquantitative ZAP-70 assay, comprising the steps of: a) staining a firstcontrol cell population with an anti-ZAP-70 antibody, wherein said firstcontrol cell population comprises mixture of cells ZAP-70⁺ cells andZAP-70⁻ cells; b) separating ZAP70⁺ cells and ZAP-70⁻ cells; c)measuring anti-ZAP-70 antibody in the separated ZAP-70⁺ cells andZAP-70⁻ cells; d) converting mean fluorescent intensity (MFI) ofanti-ZAP-70 antibody to molecules of equivalent fluorochrome (MEFL)using a standard curve and using the median MEFL as the ZAP-70 value; e)repeating steps i)-iv) using a second control cell population comprisingnormal human blood; f) repeating steps i)-iv) on a patient blood sampleif the amount of ZAP-70 in the first control cell population and secondcontrol cell population are within pre-established normal parameters. 9)The assay of claim 8 where the anti-ZAP-70 antibody comprises an Alexafluor® 647 stain. 10) The assay of claim 9, wherein the first controlcell population is a mixture of Jurkat and Ramos cells. 11) The assay ofclaim 10, wherein the ZAP-70⁺ and ZAP-70⁻ cells are electronicallyseparated by flow cytometry. 12) The assay of claim 11, wherein if thelevel of ZAP-70 in the patient B-cells is in the top 30% indicates thatthe patient has progressive chronic lymphocytic leukemia (CLL), and ifthe level of ZAP-70 in the patient B-cells is in the lower 55% indicatesthat the patent has indolent CLL. 13) A quantitative ZAP-70 assay,comprising the steps of: a) staining a mixture of T-cells and B-cellswith an anti-ZAP-70 Alexa fluor® 647 antibody, anti-CD19 antibody andanti-CD5 antibody, wherein the mixture of B-cells and T-cells is amixture of Ramos and Jurkat cells; b) separating the B-cells and T-cellsby flow cytometry using the anti-CD19 antibody and anti-CD5 antibody; c)measuring anti-ZAP-70 antibody in the separated B-cells and T-cells; d)converting mean fluorescent intensity (MFI) of anti-ZAP-70 antibody tomolecules of equivalent fluorochrome (MEFL) using a standard curve andusing the median MEFL as the ZAP-70 value; e) repeating steps i)-iv)using a second control cell population comprising normal human blood; f)repeating steps i)-iv) on a patient blood sample if the amount of ZAP-70in the first control cell population and second control cell populationare within pre-established normal parameters, wherein a ZAP-70 level inthe patient B-cells in the top 30% indicates that the patient has ashorter time to progression of chronic lymphocytic leukemia (CLL), and aZAP-70 level in the lower 55% indicates that the patient has indolentCLL.